Effects of Exposure to 17β-Estradiol (E<sub>2</sub>) and Corexit-Enhanced Water-Accommodated Fraction of Crude Oil (CWAF) <i>In Vitro</i> on Sex Determination in the American Alligator, <i>Alligator mississippiensis</i>
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Williams, Cameron Elizabeth
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Deepwater Horizon spilled over 200 million gallons of oil into the waters of the Gulf of Mexico in 2010. In an effort to contain the spill, chemical dispersants were applied to minimize the amount of oil reaching coastal shorelines. However, the biological impacts of chemically-dispersed oil are not well characterized, and there is a particular lack of knowledge concerning sublethal long-term effects of exposure. This study examined potential estrogenic effects of CWAF, Corexit 9500-enhanced water-accommodated fraction of oil, by examining its effect on estrogen receptors and sex determination in the American alligator, <i>Alligator mississippiensis</i>. The alligator exhibits temperature-dependent sex determination in which estrogen signaling is essential, and exposure to 17β-estradiol (E<sub>2</sub>) and estrogenic compounds <i>in ovo</i> during the thermosensitive period (TSP) of development can induce ovarian development at a male-producing temperature (MPT). CWAF induced transactivation up to 50% of the maximum induction by E<sub>2</sub> via <i>A. mississippiensis</i> estrogen receptors <i>in vitro</i>. To determine potential endocrine-disrupting effects of exposure directly on the gonad during TSP, gonad-adrenal-mesonephric (GAM) organ complexes were isolated from embryos one day prior to TSP and exposed to E<sub>2</sub>, CWAF or media alone <i>in vitro</i> for 8-16 days at MPT. Gonadal sex was assessed by the sexually dimorphic ratio of mRNA abundances of aromatase (<i>CYP19A1</i>) and anti-Müllerian hormone (<i>AMH</i>) using quantitative PCR (qPCR). Both CWAF and E<sub>2 </sub>exposure induced a significant increase in female ratios. GAM mRNA abundances of genes involved in the sex determination process such as Forkhead box protein L2 (<i>FOXL2</i>), sex determining region Y-box 9 (<i>SOX9</i>), and steroidogenic factor 1 (<i>SF1</i>) were also quantified using qPCR. CWAF exposure suppressed GAM mRNA abundances of <i>AMH</i>, <i>SOX9</i>, and <i>CYP19A1</i>, whereas E<sub>2</sub> exposure suppressed <i>AMH</i> and increased <i>FOXL2</i> mRNA abundances in GAM. These results indicate that the observed endocrine-disrupting effects of CWAF are not solely estrogenically mediated.